This program will use multiple approaches to identify the genetic basis of a significant subset of congenital heart defects called conotruncal and related heart defects (CTDs). We hypothesize and data suggest that a range of genetic mechanisms contribute to the etiology of these birth defects including SNPs, CNVs and rare variants, and that a multitude of approaches will help identify disease-related variants. To elucidate these mechanisms, in Aim 1 relatively unbiased genome wide analyses will be completed using a large, non- syndromic CTD cohort ascertained over the last 15 years. In particular an exact replication of our successful family-based discovery analyses will be performed, and inherited, de novo and maternal copy number variations (CNVs) that are associated with CTDs will be identified using a case-control design. Aim 2a will explore the hypothesis that the 22q11.2 deleted cohort, at-risk for CTDs, will unmask disease-related genes that also pertain to the larger population of non-deleted CTD cases. To test this hypothesis, genetic modifiers of the cardiac phenotype in the 22q11.2 deleted cohort (from Project 1) will be assessed for disease- association in the non-syndromic cohort (with matched CTDs) to replicate findings from the 22q11.2 deleted cohort and identify variants conferring disease risk in the CTD cohort. Aim 2b will translate discoveries made in mouse models (Project 3) deciphering the biological underpinnings of conotruncal development via the Tbx1 developmental pathway, into human disease. Genes and genetic pathways defined by the mouse experiments will be assessed for disease-relatedness in the non-syndromic CTD cohort. Finally, in Aim 3 large scale deep sequencing to identify rare and common genetic variants in disease-associated genes/loci identified from Aims 1 and 2 will be performed to identify the range of genetic mechanisms causing non- syndromic CTDs. The proposed studies leverage unique large syndromic and non-syndromic study cohorts to decipher the genetic basis of CTDs and apply both genome wide and candidate gene approaches. The family based model identifies both inherited and novel matemal genetic effects. Deep sequencing of associated and candidate loci will identify both rare and common disease-associated variants. RELEVANCE (See instructions): Congenital heart defects are the most common, serious birth malformation affecting approximately 1 in 200 live births. Despite their prevalence and public health implication, their etiology remains poorly understood. These studies will elucidate the genetic basis for a subset of these malformations so that novel therapeutic and preventive strategies can be designed, and clinical management and outcomes improved.